Pyrroline based compounds

ABSTRACT

A 1-PYRROLINE-2-ACRYLAMIDE, WHICH IS SUITABLE FOR USE AS AN ANTIBIOTIC (HEREIN REFERRED TO AS PYRACCRIMYCIN A) IS PRODUCED BY CULTIVATION OF STREPTOMYCES ERIDANI N. SP. ATCC 21619 AND CAN BE ISOLATED FROM THE FERMENTATION MEDIUM BY EXTRACTION. THE ANTIBIOTIC SUBSTANCE IS ACTIVE AGAINST VARIOUS GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA. THEY3 DIH DRO DERIVATIVE OF 1-PYRROLINE-2-ACRYLAMIDE IS ALSO DISCLOSED.

United States Patent O 3,798,130 PYRROLINE BASED COMPOUNDS Carolina Coronelli, Giangualberto Gallo, and Graziella Beretta, Milan, Italy, assignors to Gruppo Lepetit S.p.A., Milan, Italy N Drawing. Original application Jan. 28, 1971, Ser. No. 110,755, now Patent No. 3,737,439. Divided and this application Nov. 7, 1972, Ser. N 0. 304,355

Int. Cl. C12k N00 US. Cl. 195-80 R 4 Claims ABSTRACT OF THE DISCLOSURE A 1-pyrroline-2-acrylamide, which is suitable for use as an antibiotic (herein referred to as pyracrimycin A) is produced by cultivation of Streptomyces eridani n. sp. ATCC 21619 and can be isolated from the fermentation medium by extraction. The antibiotic substance is active against various Gram-positive and Gram-negative bacteria. The dihydro derivative of l-pyrroline-2-acrylamide is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION This is a division of application Ser. No. 110,755, filed Jan. 28, 1971, now US. Pat. No. 3,737,439.

BRIEF SUMMARY OF THE INVENTION This invention is directed to novel compounds based on 1-pyrroline-2-acrylamide, which are particularly suitable for use as antibiotic substances and is inclusive of a method for preparing the same. l-pyrroline-Z-acrylamide (hereinafter referred to at times as pyracrimycin A) is a white crystalline substance having characteristic properties such as melting point, infrared and ultraviolet absorption spectra, proton magnetic resonance and mass spectra. On the basis of these data together with the elemental analysis and the molecular weight, l-pyrroline-2-acrylamide can be assigned the following structural formula The pyracrimycin A form of 1-pyrroline-2-acrylamide can be prepared by cultivation of Streptomyces eridani n sp. ATCC 21619 in an aqueous medium and can be isolated from the medium together with another metabolite hereinafter referred to as pyracrimycin B which is another derivative of 1-pyrroline-2-acrylamide. The separation of pyracrimycin A from pyracrimycin B, which does not possess antibiotic activity, can be effected by treating the crude mixture of the two metalbolites with hot chlorinated lower hydrocarbons of from 1 to about 4 carbon atoms such as, for instance, chloroform, in which pyracrimycin A is substantially insoluble.

In the preparation of pyracrimycin A the organism is cultivated under aerobic conditions in aqueous nutrient medium suitable for the growth of said organism, for a period of from about 20 to about 120 hours at a temperature of from about 20 to about 45 C., the medium containing a source of carbon, at source of nitrogen and inorganic salts. The cultivation is continued under conditions which favor the growth of the microorganism for a time suflicient to produce substantial antibotic activity. For instance, a paper disc agar plate assay with E. coli may be used to control the antibiotic level. Pyracrimycin A thus produced can be isolated by conventional procedures such as removal of mycelium by filtration followed by extraction with an organic solvent in which the 1-pyrroline-2-acrylamide materials are soluble and which is immiscible with the aqueous medium. A crude mixture 3,798,130 Patented Mar. 19, 1974 of pyracrimycin A and B may be obtained by adding a large excess of petroleum ether to the organic extract after having washed with alkaline water and concentrated the solution. The crude mixture of the two metabolites is then purified by suspending in methanol, filtering off the insoluble impurities concentrating the filtrate and chilling the solution until crystallization is complete. A separation of the two products is achieved according to the afore-mentioned treatment with a chlorinated organic solvent. The antibiotic substance of the invention is useful against Gram-positive and Gram-negative bacteria. The following Table I illustrates the antimicrobial activity of the separated l-pyrroline-Z-acrylamide identified as pyracrimycin A.

TABLE I Antimicrobial activity or pyracrimycin A Minimal inhibitory Microorganism test: concentration, 7/ ml. Staphylococcus aureus 209 P 20 Stephylococcus aureus Tour 20 Streptococcus hemolyticus C 203 20 Diplococcus pneumoniae UC 41 l0 Candida albicans SKF 2270 One of the most important characteristics of this new antibiotic substance consists in the fact that in the in vitro tests practically no development of resistant strains is observed. This property is very significant where a topical application of an antibiotic substance is planned since in this case a widespread use of the substance has to be taken into consideration. The widespread use of an antibacterial substance may lead not only to emergence of resistant mutants within an individual patient but also also to the spread of resistant strains. It is therefore evident that a substance which inhibits development of resistant strains oflfers undoubted advantages particularly when used for widely dilfused topical applications. In our case, the antimicrobial substances of the invention may be easily compounded into ointments with the usual pharmaceutical carriers and binders such as lanolin, petrolatum, liquid petroleum, water, emulsifying and thickening agents. 1-pyrroline-2-acrylamide can be hydrogenated in lower alkanol solution, i.e. those having from 1 to about 4 carbon atoms, with metal borohydrides, particularly an alkali metal borohydride, such as, for instance, sodium borohydride at a temperature ranging from about 0 to about 30 C. for a period of from about 2 to about 18 hours, to a dihydro derivative which on the basis of the physical data corresponds to the structure of 2-pyrro1idinacrylamide 3 4 Also, this compound when prepared from pyracrimycin Production and properties of l-pyrroline-Z-acryl- A is endowed with antimicrobial activity. In a represenamide (pyracrimycin A) tative in vitro test it inhibits the growth of Escherichia For roducin the invention antibioti substanc the col: strains at a concentration of 50 'y/ml. p g c 6 strain Streptomyces eridani n. sp. ATCC 21619 is aero- AH V bically cultivated in an aqueous nutrient medium until DET ED DESCRIPTION OF THE INVENTION substantial antibiotic activity is present in the same medi- (1) Description of Streptomyces eridamurn. As an example, a vegetative medium for shake flask ATCC 21619 culture may have the following composition in gr./l.

I Beef extract 5 Streptomyces erza'am may be grown on a variety of Yeast extract 5 standard media according to Gottlieb and Shirling, Int- Pgptone, 5 Journal of Systematic Bacteriology 16, 313, 1966. Som Enzymatic casein hydrolysate 3 other media may also be useful as it appears from Table Dextrose II which reports the cultural and physiological characs di hl id 1.5 teristics of the producing strain. 10 Tap Water q.s. to 1000 ml.

TABLE II [Cultural and physiological characteristics of Streptomyces cridam, n. Sp. ATTC 21619] Physiological Culture medium Vegetative mycehum Aerial mycehum Soluble pigment characteristics Oat meal agar Ggg g ga 1 1, smooth sur- Whitish in traces Cream to light amber--- Medium 11. 2 (Gottlieb and Shirling) Good growth slightly Absent Amber brown wrinkled, amber. 0ath1rna1)agar (Medium n. 3 Gottlieb and Moderate growth, smooth White, velvety, Absent r rig surface, hyaline. not much abundant. Glycerol asparagine agar (Medium n. 6 Growth good, smooth sur- Absent (In Gottlieb and Shirling). face, cream. Hickey and Tresners agar Good growth, slightly do Deep amber brown wrinkled, brown.

Bennett's agar Good growth, wrinkled surface, amber brown. Czapek glucose agai- Moderate growth, smooth surface, straw. Glucose asparagine agar Good growth, smooth surdo Traces, cream face with wax aspect, cream. Nutrient agar Growth scarce, thin smooth do Amber surface, amber Potato agar Good growth, shghtly Whitish in traces Light brown {)vnnkled surface, light rowri. Starch agar (Medium n. 4 Gottlieb and Moderate growth, smooth Absent Straw Good hydrolysis.

Shirling). surface, straw. Pepton-yeast extract iron agar (Medium n. Moderate growth, smooth rln Pr d ti f H 8,

6 Gottlieb and Shirling). surface, brown.

Tyrosine agar (Medium 11. 7 Gottheb and Good growth, wrinkled surdo Black-brown at the edges Tyrosinase reaction posi- Shirhng). face, black brown. of the growth, brown tive, strong production in the medium. of melanoid pigment. Ca malate agar Good growth, smooth surrln Absent Strong digestion of Ca face, straw. malate. Gelatin- (in Liquefaction. N trate broth Dark brown Nitrate reduction. Litmus milk Brown ring Absent N o peptonization, no

. coagulation.

Skim milk agar Good growth, smooth sur- Absent Brown, not very soluble No hydrolysis of casein.

face. in the medium.

The most convement temperature for development of Th fl k are h k fo about 43.50 hours at a m the 601011168 fangfis from about to about and perature of about 28-30 C. on a reciprocating shaker. more particularly from a u 8 to a ut 37 C. Such a culture 18 used as an inoculum to be added to the Aeflal mycehum 1S Produced y erldanl y 011 Oat vegetative medium of the fermentors. To this purpose, meal and potato agar. On the latter the aerial mycehum m a f t 4 1, f a medmm havlng th following produced is scanty and no sporulation is observed; on oat iti i ,/1, meal agar the aerial mycehum 15 White velvety with long Beef extract 4 flexous and branched hyphae having a diameter of about 1.3 The aerial hyphae produce sporophores in fairly closed spirals different in their lengths. The spores have diameters of about 1-1.3,a x 1.3-1.6,u. The following Table III reports the test for utilization of carbon sources Peptoiie NaCl Yeast extract Soybean meal according to Pridham and Gottlieb, Journal Bacteriology, D extrose CaCO 56, 107, 1948.

Water (1.8. t0 ml.

is inoculated with an 8% of the shake flask culture and Utlhzatlon 3 355; g gg g g g g maintained under agitation of 800 r.p.m. and aeration of Carbon Source. Growth l./v./v./min. for about 24 hours at a temperature of Sucrose l about 28-30 C. The further fermentation operations are carried out in fermentors containing 10 l. of a medium having practically the same composition as above with lnositol g 0f dBXtIOSE. aid medium is inoculated with 1 Raft-mo liter f he ab ve vegetative medlum cul ture and 1S mam- Mannitol tamed at about 28-30 C. under agitation of 800 r.p.m. Fructose nd a ra i n f 1./v./v./m1n. At intervals the antlblotic Rhamnose actlvlty assayed p p r d1 agar plateusrng E. coli c llul as test mi r organism. The maximum actlvlty is reached Glucose (positive control) after 48 hours of nta i n. The fermented broth is filtered with the aid of 2% Hyflo-Super Cel and the gi fifig j utuzatmn' mycelial cake is discarded. The filtrate, after the addition of 20% of NaCl is extracted twice with one-half its volume of butanol, the combined extracts are washed with small volume of slightly alkaline water and concentrated to one fifth of the original volume. A precipitation of inorganic salts, containing less than 1% of the pyracrimycins is obtained by cooling a few hours at 4 C. the concentrated solvents; the salts are filtered off and the solution concentrated again to a small voume. A crude mixture of pyracrirnycin A ad B is obtained by adding a large excess of petroleum ether to the concentrated extracts. The crude material has about 30-35% purity, pyracrimycin A and B being present in roughly one to one proportion. The product is suspended in hot methanol grs., 500 ml.) and filtered from insoluble matter. The solution is treated with charcoal to remove impurities, concentrated until crystallization starts and chilled until crystallization is complete; both metabolites crystallize in mixture giving a product with about 95% total purity. A separation of the two metabolites is achieved by treat ment of the product with chloroform on water bath for one hour. Pyracrimycin A, containing only 12% of B, is collected and pyracrirnycin B crystallizes after concentration of the chloroform solution to a small volume. A final crystallization from methanol gives the pure product.

Pyracrirnycin A is a white crystalline substance M.P. 215-2l6 C. The product resulted to be unitary on paper and thin layer chromatography; the Rf values obtained with different solvent systems are reported in Table IV.

Paper chromatography on Wh. l, antibiotic visualized on agar plates seeded with S. aureus.

TLC on silica gel plates, spot detected under UV light.

The found microanalytical data (C, 60.99%; H, 7.50%; N, 20.20%) are consistent for the molecular formula C H N O (theoretical values: C, 60.85%; H, 7.30%; N, 20.27%) with a molecular weight 138.17. The product has slightly basic character, is almost insoluble in the common organic solvents, with the exception of dimethylsulfoxide and dimethylformamide and slightly soluble in dilute acids, methanol and lower alkanols.

The infrared spectrum in mineral oils shows characteristic bands at 3250, 3050, 1670, 1620, 1590, and 972 cm.- which are consistent with the assigned structure 1-pyrroline-2-acrylamide. The U.V. spectrum in methanol solution shows a maximum at 235 m (e 22,000).

Potentiometric titration in a 1:4 mixture of water and methyl Cellosolve with 0.1 N hydrochloric acid shows a basic ionizable function having a pK value of 5.4.

The proton magnetic resonance spectrum in DMSO-d,; solution is recorded with a Varian A-60 spectrometer at 60 megacycles in the customary manner using tetramethylsilane as internal standard. The principal characteristic absorption peaks occurs at the following frequencies expressed in 6 units 1.87 (multiplet), 2.67 and 3.90 (two triplets), 6.44 and 7.16 (two doublets).

The mass spectrum of pyracrimycin A is recorded with a mass spectrometer Hitachi-Perkin Elmer RMU-6L and confirms the molecular weight 138.17 by the M+ peak at 138 m/e units.

Preparation and properties of 2-pyrrolidinacrylamide (dihydropyracrimycin A) To a solution of 0.5 gram of pyracrimycin A in 300 ml. of dry ethanol cooled on ice-bath, 0.25 gram of sodium borohydride is added. The solution is kept at about 4 C. for two hours and at room temperature for 12 hours under stirring. After decomposing the excess of NaBH, with 10% HCl, the ethanol solution is diluted with water and then evaporated, under reduced pressure. The residual aqueous solution after alkalization with 10% sodium carbonate is thoroughly extracted with ethyl acetate. The extracts, after anhydrification and concentration to dryness, give 0.1 gram of a highly hygroscopic product which is dihydropyracrimycin A. The compound shows the following properties:

Analysis.--Found for C H N O (percent): C, 59.45; H, 8.80; N, 19.60. Calculated (percent): C, 59.98; H, 8.63; N, 19.98.

Molecular weight confirmed by mass spectrum: 149.19.

Thin-layer chromatography on silica gel plate Rf=0.6 (ethyl acetatezacetic acidrwater 3:121).

Characteristic band of infrared spectrum in Nujol expressed in cm.- 33 00, 3130, 1680, 1620, 990.

Potentiometric titration in water with 0.1 N HCl gives a pK value of 9.4.

The P.M.R. spectrum in DMSO-d shows the following significant peaks expressed in 5 units which confirm the reduction of the carbon-nitrogen double bond in the 2-pyrroline moiety: 5.92 (doublet); 6.55 (doublet of doublets).

We claim:

1. A process for the production of l-pyrroline-Z-acrylamide exhibiting antibacterial activity which comprises cultivating Streptomyces eridani n. sp. ATCC 21619 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium, and recovering said l-pyrroline-Z-acrylamide from said medium.

2. The process as defined in claim 1 wherein the cultivating is carried out under submerged aerobic conditions for a period of from about 20 to about hours at a temperature of from about 20 to about 45 C.

3. The process as defined in claim 1 and including the steps of filtering the aqueous medium containing said 1-pyrroline-2-acrylamide, extracting the filtrate with a solvent immiscible with water thereby providing an extract of said 1-pyrroline-2-acrylamide, and treating said extract with a chlorinated lower hydrocarbon, and recovering a l-pyrroline-Z-acrylamide exhibiting antibacterial activity.

4. The process as defined in claim 1 and including the step of reacting said 1-pyrroline-2-acrylamide with a metal borohydride in a lower alkanol solution at a temperature ranging from about 0 to about 30 C. for a period of from about 2 to about 18 hours to produce 2-pyrrolidinacrylamide.

References Cited UNITED STATES PATENTS 3,364,115 1/1968 Mason et al 80 R 2,928,844 3/1960 Boer et al. 195-80 R A. LOUIS MONACELL, Primary Examiner R. I. WARDEN, Assistant Examiner 

